The discovery of substances that control the growth of animal cells, especially human cells, and the mechanism by which they work is currently one of the major focuses of biomedical research concerned with tissue repair and wound healing. Fibroblast growth factors (FGFs), mitogens for various cell types including many cells of mesodermal origin, have been identified and it has been suggested that they may induce mitosis which will result in tissue repair. Fibroblast mitogenic activity was first observed with extracts of tissue from the central nevous system. Brain-derived fibroblast mitogens were first described by Trowell et al., J. Exp. Biol. 16: 60-70 (1939) and Hoffman, Growth 4: 361-376 (1940). It was subsequently shown that pituitary extracts also had potent mitogenic activity for fibroblastoid cells, Amelin, Proc. Natl. Acad. Sci. USA 70: 2702-2706 (1973). Partial purification of both brain and pituitary fibroblast growth factor revealed mitogenic activity for a variety of cell types of differentiated cells including vascular endothelial cells, Gospodarowicz et al., Natl. Cancer Inst. Monogr. 48: 109-130 (1978). Fibroblast growth factor was originally thought to be a single peptide derived from the limited proteolysis of myelin basic protein. It has recently been shown that FGF exists in two forms, acidic FGF (aFGF) and basic FGF (bFGF), and both forms can be isolated and purified from mammalian brain, Thomas and Gimenez-Gallego, TIBS 11: 81-84 (1986). Numerous cell types respond to stimulation with either purified aFGF or bFGF to synthesize DNA and divide, including primary fibroblasts, vascular and corneal endothelial cells, chondrocytes, osteoblasts, myoblasts, smooth muscle, glial cells and neuroblasts, Esch et al., Proc. Natl. Acad. Sci. USA 82: 6507-6511 (1985); Kuo et al., Fed. Proc. 44: 695 (1985); Gensburger et al., C.R. Acad. Sc. Paris 303: 465-468 (1986). Pure bovine brain-derived aFGF not only acts as a potent mitogen for vascular endothelial cells in culture but also induces blood vessel growth in vivo, Thomas, et al. Proc. Natl. Acad. Sci. USA 82: 6409-6413 (1985). The mitogenic activity of purified aFGF can also be used to promote wound healing, Thomas, U.S. Pat. No. 4,444,760.
Acidic fibroblast growth factor was originally purified to homogeneity from bovine brain based on its mitogenic activity for BALB/c 3T3 fibroblasts, Thomas et al., Proc. Natl. Acad. Sci. USA 81: 357-361 (1984). This brain-derived growth factor has been repurified and renamed in multiple laboratories based both on its: mitogenic activity for vascular endothelial and astroglial cells (endothelial cell growth factor and astroglial growth factor 1), source (retinal-derived growth factor, eye-derived growth factor II, and perhaps brain-derived growth factor), and binding to heparin-Sepharose (class 1 heparin-binding growth factor or heparin-binding growth factor alpha), Thomas and Gimenez-Gallego TIBS 11: 81-84 (1986). The amino acid sequence of bovine aFGF has been determined, recognized to be highly homologous to basic FGF and related to the fibroblast mitogens interleukin 1-alpha and 1-beta, Gimenez-Gallego et al., Science 230: 1385-1388 (1985). The complete amino acid sequence of human aFGF has been determined from the purified protein, Gimenez-Gallego et al., Biochem. Biophy. Res. Comm. 138: 611-617 (1986), and from the gene, Jaye et al., Science 233: 541-545 (1986).
Native aFGF purified from brain or recombinant-derived aFGF (r-aFGF) requires the co-administration of heparin to optimally stimulate Balb/c 3T3 fibroblasts and vascular endothelial cells in culture. Human brain-derived and recombinant aFGF are only about 1% to 5% as active on these cells in culture in the absence of heparin compared to optimal activity in the presence of heparin. While the doses required for maximal aFGF activity are relatively low, it might be desirable to administer aFGF with no heparin since heparin could conceivably elicit detrimental side effects. Pure human aFGF, in addition to the standard conditions that destroy the activity of most proteins, extremes of heat, pH and the presence of proteases, is also labile to lyophilization and oxidation. The pure aFGF becomes cross-linked through intrachain or interchain disulfide bonds by oxidation and can be recovered in active form by disulfide reduction with 20 mM dithiothreitol. Heparin can inhibit intermolecular disulfide bond mediated aggregation of aFGF. This heterogeneous glycosaminoglycan has also been noted to stabilize aFGF from heat denaturation and proteolytic degradation by trypsin. Consequently, either exogenous or endogenous heparin is required for the in vivo activity associated with tissue repair. The present invention provides unique mutated forms of recombinant-derived aFGF which have an increased biological activity in the absence of heparin compared to native aFGF.